Redeployment of a conserved gene regulatory network during Aedes aegypti development

Author’s Accepted Manuscript Redeployment of a conserved gene regulatory network during Aedes aegypti development Kushal Suryamohan, Casey Hanson, Emily Andrews, Saurabh Sinha, Molly Duman Scheel, Marc S. Halfon www.elsevier.com/locate/developmentalbiology

PII: DOI: Reference:

S0012-1606(16)30186-5 http://dx.doi.org/10.1016/j.ydbio.2016.06.031 YDBIO7176

To appear in: Developmental Biology Received date: 7 April 2016 Revised date: 13 June 2016 Accepted date: 20 June 2016 Cite this article as: Kushal Suryamohan, Casey Hanson, Emily Andrews, Saurabh Sinha, Molly Duman Scheel and Marc S. Halfon, Redeployment of a conserved gene regulatory network during Aedes aegypti development, Developmental Biology, http://dx.doi.org/10.1016/j.ydbio.2016.06.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Redeployment of a conserved gene regulatory network during Aedes aegypti development Kushal Suryamohan1,2, Casey Hanson3, Emily Andrews4, Saurabh Sinha3, Molly Duman Scheel4,5, Marc S. Halfon1,2,6,7* 1

Department of Biochemistry, University at Buffalo-State University of New York, Buffalo, NY

2

NY State Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY

3

Department of Computer Science, University of Illinois Urbana-Champaign, Champaign, IL

4

Indiana University School of Medicine, Department of Medical and Molecular Genetics, South Bend, IN 5

University of Notre Dame, Eck Inst. for Global Health and Department of Biological Sciences, South Bend, IN 6

Department of Biological Sciences and Department of Biomedical Informatics, University at Buffalo-State University of New York, Buffalo, NY 7

Department of Molecular and Cellular Biology and Program in Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY

*

Correspondence author. Department of Biochemistry. University at Buffalo-State University of

New York. 701 Ellicott St. Buffalo, NY 14203. Tel.: 716-829-3126. [email protected]

Abstract Changes in gene regulatory networks (GRNs) underlie the evolution of morphological novelty and developmental system drift. The fruitfly Drosophila melanogaster and the dengue and Zika vector mosquito Aedes aegypti have substantially similar nervous system morphology. Nevertheless, they show significant divergence in a set of genes co-expressed in the midline of the Drosophila central nervous system, including the master regulator single minded and downstream genes including short gastrulation, Star, and NetrinA. In contrast to Drosophila, we find that midline expression of these genes is either absent or severely diminished in A. aegypti. Instead, they are co-expressed in the lateral nervous system. This suggests that in A. aegypti this “midline GRN” has been redeployed to a new location while lost from its previous site of activity. In order to characterize the relevant GRNs, we employed the SCRMshaw method we previously developed to identify transcriptional cis-regulatory modules in both species. Analysis of these regulatory sequences in transgenic Drosophila suggests that the altered gene

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expression observed in A. aegypti is the result of trans-dependent redeployment of the GRN, potentially stemming from cis-mediated changes in the expression of sim and other as-yet unidentified regulators. Our results illustrate a novel “repeal, replace, and redeploy” mode of evolution in which a conserved GRN acquires a different function at a new site while its original function is co-opted by a different GRN. This represents a striking example of developmental system drift in which the dramatic shift in gene expression does not result in gross morphological changes, but in more subtle differences in development and function of the late embryonic nervous system. Key words: enhancer discovery, gene regulatory networks, GRN, Drosophila melanogaster, Zika vector mosquito, Aedes aegypti, ventral midline, Central Nervous System (CNS) development, evolution of regulatory networks, developmental system drift, neofunctionalization.

Introduction Metazoan development proceeds through the activity of tightly coordinated gene expression programs, each governed by a specific Gene Regulatory Network (GRN). GRNs consist of the transcription factors (TFs) and signaling pathways that mediate downstream developmental events and critically, the cis-regulatory modules (CRMs) that control spatio-temporal patterns of gene expression. Phenotypic diversity in the animal kingdom has been postulated to be largely driven by changes in GRN structure and function (Carroll et al., 2005; Davidson, 2006). Such changes can occur at either or both of two levels. At the trans level, TFs can be added to or removed from the network, while at the cis level, individual CRMs can gain or lose the ability to bind specific TFs. The resulting changes in GRN structure can lead to loss, cooption, or neofunctionalization of GRNs or GRN sub-circuits. The effects, viewed from the perspective of the GRN, can range from minor (if they occur near the terminal branches of the network) to dramatic phenotypic alterations (if they occur toward the top of the GRN regulatory hierarchy) (Gompel et al., 2005; Peter and Davidson, 2011; Prud'homme et al., 2006; Prud'homme et al., 2011; also reviewed in Rebeiz et al., 2015; Wittkopp et al., 2002). There are also striking examples of conserved phenotypic outcomes generated by non-homologous genes and presumably, widely diverged GRNs, a phenomenon which when viewed from this perspective is referred to as “developmental system drift” (True and Haag, 2001) or “phenogenetic drift” (Weiss and Fullerton, 2000). By exploring GRN changes and the resulting phenotypes in organisms of different degrees of evolutionary divergence, we can link genotypic variation to

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phenotypic outcomes and gain insight into the mechanisms governing convergent and direct evolution, developmental system drift, the emergence of morphological novelties, and the robustness of phenotypes in the face of regulatory sequence turnover. However, despite a growing number of clear examples of GRN co-option and neofunctionalization (Glassford et al., 2015; McCauley et al., 2010; Prud'homme et al., 2006; Rebeiz et al., 2011; and reviewed in Rebeiz et al., 2015), detailed GRN-level studies have been limited owing to a lack of known CRMs, TF interactions, and gene expression patterns for related organisms. The Drosophila melanogaster embryonic central nervous system (CNS) is a well-established system for studying the molecular and genetic mechanisms governing cell fate specification. The overall CNS structure is common to all insects, with a ladder-like assembly of longitudinal and midline-crossing axon tracts and a similar arrangement of neuronal stem cells (neuroblasts) and neuroblast progeny (Duman-Scheel and Patel, 1999). However, it is becoming increasingly clear that this highly conserved structure may mask a substantial amount of developmental system drift, with widespread differences in patterns of gene expression within seemingly homologous neuronal cells suggesting that significant changes in the underlying GRNs may exist despite the morphologically similar outcomes (Biffar and Stollewerk, 2014). One such change appears to be in the GRN regulating development of the ventral midline. Like its vertebrate counterpart the floor plate, the midline of the insect nerve cord is a specialized structure critical for normal development and an important source of inductive signals and axon guidance molecules (Tessier-Lavigne and Goodman, 1996). In Drosophila, the midline cells are derived from the mesectoderm, two single-cell wide rows abutting the presumptive mesoderm of the blastoderm embryo. During gastrulation, these cells are brought together to form the ventral midline. As development proceeds, they proliferate and differentiate into the midline glia and a small set of midline neurons (Wheeler et al., 2006). The key regulator of midline development is the bHLH-PAS transcription factor Single minded (Sim) (Crews et al., 1988). sim expression marks the mesectoderm, and Sim directly activates midline-expressed genes while indirectly repressing lateral CNS genes (Estes et al., 2001; Nambu et al., 1990). sim expression persists in all midline cells until germband retraction, after which it is maintained in most although not all of the lineage (see also Results). Sim binding sites have been identified in almost all known midline CRMs, and Sim binding contributes to midline gene expression in all cases tested to date (Pearson and Crews, 2014). Although the overall structure of the GRN regulating midline development remains to be elucidated, sim appears to be a “master regulatory gene” that sits at

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the head of this GRN and is directly or indirectly responsible for many of the subsequent developmental events. The pivotal role of sim in midline development is underscored by the fact that in all insects studied to date, sim expression is found at the ventral midline at early and mid-embryonic stages. Intriguingly, however, the width of the midline expression is variable, ranging from 1-2 cells in D. melanogaster, to ~3 cells in the beetle Tribolium castaneum, to 5-6 cells in the honeybee Apis mellifera (Zinzen et al., 2006). Analysis of the CRMs responsible for early sim expression indicates that different TFs activate sim in different insects (Zinzen et al., 2006). For instance, in A. mellifera, sim appears to be regulated primarily by Twist, whereas in the mosquito Anopheles gambiae, sim is regulated by the Notch pathway instead. While the more recently evolved D. melanogaster regulates sim via both Twist and Notch, sim regulation in the related Drosophilids D. pseudoobscura and D. virilis resembles the Notch-based regulation seen in A. gambiae. Downstream genes regulated by Sim, such as rho, slit, and sog, follow the pattern of sim expression (wide in bees, narrow in flies), demonstrating how cis-regulatory changes for a key TF (Sim) lead to a trans-regulatory change affecting multiple downstream GRN members. As noted above, however, despite these changes there are few notable differences in the final CNS structure; CNS development appears to be robust to the early GRN changes. We describe here a dramatic GRN change in which there is an apparent relocation of a GRN from the midline to the lateral CNS in late embryonic stages in the dengue and Zika vector mosquito Aedes aegypti, but again with seemingly minimal consequences for development. We find that in A. aegypti, unique among the insects studied to date, sim and a number of additional genes expressed in the Drosophila midline are expressed in the lateral CNS rather than the midline in the late embryo. Despite this change, both D. melanogaster and A. aegypti retain what appear to be structurally and functionally homologous CNS midlines (Clemons et al., 2011; Simanton et al., 2009), including with respect to genes expressed in differentiated midline cells. We identify CRMs for several of the affected A. aegypti genes and test their function in transgenic Drosophila, where they drive a D. melanogaster-like pattern of gene expression. These data suggest that trans-regulatory mechanisms are likely to be responsible for the shift in gene localization. We further demonstrate that a shift in sim expression pattern alone is not sufficient to account for the changes we observe in downstream gene expression. Our work illustrates a striking example of developmental system drift in which a GRN has been spatially

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redeployed, with significant changes in gene expression patterns, yet where the developmental outcomes remain largely unchanged.

Materials and Methods Selection of candidate CRMs We developed in previous work the SCRMshaw (for “Supervised Cis-Regulatory Module prediction”) approach for accurate prediction of enhancers in Drosophila (Kantorovitz et al., 2009; Kazemian et al., 2011a). Briefly stated, SCRMshaw uses a training set of Drosophila enhancers defined by a common functional characterization (e.g., ‘salivary gland’, ‘midgut’, ‘testes’) to build a statistical model that captures their short DNA subsequence (k-mer) count distribution (see cited papers for details). An appropriate set of background sequences (e.g., random non-coding sequences) is also used in this training phase. The trained model is then used to score overlapping 500 bp windows in the “target genome,” and the highest peaks in the resulting score profile are predicted to be enhancers. We showed in Kazemian et al. (2014b) that the target genome could be not just the D. melanogaster genome but also that of another holometabolous insect. In this work, SCRMshaw predictions were generated for the D. melanogaster and A. aegypti genomes using the dm3/release 5 and AaegL3.3 genome releases, respectively. Briefly, candidates from each species were selected on the basis of the nearest annotated gene for each predicted CRM. Ten CRM predictions from A. aegypti were chosen whose nearest annotated A. aegypti gene (within 25 Kb) had a known D. melanogaster ortholog with expression in the CNS midline (Table S2). For D. melanogaster, thirteen CRM predictions near annotated genes (within 20 Kb) with known midline expression were picked for in vivo validation (Table S1). Fly Stocks Ectopic gene expression was induced with the Gal4-UAS bipartite expression system (Brand and Perrimon, 1993) using a prd-Gal4 driver line (Bloomington Drosophila stock center BL1947) and either UAS-sim (P(UAS-sim.X)2 (Xiao et al., 1996), Bloomington Drosophila stock center BL9581) or UAS-Nintra (Kidd et al., 1998). Reporter gene constructs on the second chromosome were crossed into the prd-Gal4 background prior to crossing the Gal4 and UAS lines together. Embryos from these crosses were collected at approximately stage 12 of embryogenesis.

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Mosquito Rearing and Embryo Fixation Aedes aegypti Liverpool- IB12 (LVP-IB12) and Culex quinquefasciatus Johannesburg (JHB) strains, which correspond to the strains used in the genome sequencing efforts (Nene et al., 2007; Arensburger et al., 2010), were used in these studies. Both strains were reared as previously described (Clemons et al., 2010a), except that an artificial membrane blood feeding system was employed. Mosquito embryos were fixed as described (Clemons et al., 2010b). In situ hybridization and Immunohistochemistry For A. aegypti embryo in situ hybridizations, digoxygenin-labeled riboprobes (~500 bp in size) corresponding to the following A. aegypti genes were synthesized as described (Patel, 1996): A. aegypti sim (AAEL011013), NetA (AAEL003024), sog (AAEL005627), S (AAEL005704), and Cqu sim (CPIJ009948). In situ hybridization (Haugen et al., 2010) was performed as described. T. cas and A. melanogaster embryos were generously provided by Yoshi Tomoyasu (Miami University) and Gene Robinson (University of Illinois Urbana-Champaign), respectively. T. cas and A. melanogaster in situ hybridization was performed as described in (Dearden et al., 2009; Schinko et al., 2009). sim probe sequences were obtained from (Zinzen et al., 2006) and (Cande et al., 2009) and generated by PCR from genomic DNA. In situ hybridization for D. melanogaster genes was performed using standard protocols (Tautz and Pfeifle, 1989). The following clones were used to generate digoxygenin-labeled riboprobes: NetA (RE11206); runt (FI01105); sim (RE54280); sog (LP09189). Immunohistochemistry was performed using standard methods. Primary antibodies used were mouse anti-LacZ (1:500; Abcam) and guinea-pig anti-Sim (1:200; gift of Steve Crews). For some embryos, Fluorescein Tyramide (Perkin Elmer TSA kit) was used for signal amplification (1:500). Fluorescent images were acquired using a Leica SP2 confocal microscope. Reporter Constructs and Transgenic Drosophila Genomic sequences were amplified by PCR from genomic DNA from the appropriate species, cloned into pJet1.2blunt (Fermentas), and confirmed by sequencing. Primer sequences are provided in supplementary Table S3. The putative CRM sequences were subcloned into plasmid pBlueRabbit, a øC31-enabled Drosophila transformation vector containing the LacZ gene under the control of a minimal hsp70 promoter (Housden et al., 2012), with the exception

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of the Aaeg sim 8 kb fragments, which were cloned in pGreenRabbit (Housden et al., 2012). Transgenic flies were produced by Genetic Services Inc. (Cambridge, MA) and by Rainbow Transgenic Flies (Camarillo, CA) by injection into lines attP2 or attP40.

Results A unique pattern of single minded expression in the CNS of Aedes aegypti embryos In Drosophila, the first transcripts of sim are detected in the blastoderm embryo and are restricted ventrally to two rows of cells on each side of the presumptive mesoderm, together forming the mesectoderm. Following gastrulation these two rows converge at the embryonic ventral midline (Fig. 1A), where sim expression is maintained by autoregulation through germband extension (Fig. 1B) (Muralidhar et al., 1993; Nambu et al., 1991; Wharton et al., 1994). From germband retraction (stage 12) through to late embryogenesis, sim continues to be expressed in the majority of midline cells, including the midline glia, the iVUMs, H-cell sib, and the progeny of the midline neuroblast (Wheeler et al., 2008). We refer to these three stages of expression as “mesectoderm,” “midline primordium,” and “late midline,” respectively. Although the mechanisms responsible for the late midline stage of sim expression have not been definitively established, it has been proposed to be dependent on Notch signaling and possibly autoregulation (Pearson and Crews, 2014). In all insects studied to date, sim expression is found at the ventral midline through the midline primordium stages, although the width of the midline expression is variable (from 1-2 cells in D. melanogaster to 5-6 cells in the honeybee A. mellifera) (Zinzen et al., 2006). In A. mellifera there is also segmentally-repeated expression that has been reported to be clusters of lateral CNS cells at the midline primordium stage (Fig 1E, arrowheads) (Zinzen et al., 2006). However, we note that this expression resembles that seen at a similar stage in D. melanogaster, which has been shown to be mesodermal muscle progenitor cells that sit on the surface of the nerve cord and subsequently migrate to the periphery to form ventral oblique muscle fibers (Fig. 1C, arrowheads, see also Lewis and Crews, 1994). Similar expression is observed in D. virilis (KS, unpublished observations). As late-stage A. mellifera embryos also express sim in the ventral oblique muscles—and not in the lateral CNS (Fig. 1F, G)—the transient “lateral CNS” clusters are likely in fact to be early muscle precursors. We have not so far observed similar

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mesodermal expression in the mosquitoes Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus, the beetle Tribolium castaneum, or the jewel wasp Nasonia vitripennis at comparable stages (MDS, KS unpublished observations; J. Lynch, pers. commun.). However, given the weak and transient nature of the expression in flies and bees, we cannot fully rule out the possibility of limited muscle progenitor expression in these other insects as well. In all of these insects save A. aegypti, sim expression persists in and (with the exception of the noted muscle progenitors) is confined primarily to a subset of midline cells throughout the late midline stage (Fig. 1D, F, J, K and data not shown). Strikingly, however, the late midline stage pattern of sim expression is different in A. aegypti. Despite robust midline expression at the midline primordium stage continuing through germband extension (Fig. 1H and data not shown), at the late midline stage following germband retraction A. aegypti sim expression is highly diminished or possibly absent in midline cells. At the same time, however, and unique among insects so far examined, sim is detected at high levels in late-stage lateral CNS clusters (Fig. 1I).

Additional “midline” genes are also expressed laterally rather than at the midline in germbandretracted A. aegypti embryos Given the surprising finding that A. aegypti sim is expressed laterally rather than at the midline in late-midline stage embryos, we looked at the expression of additional genes that are expressed either primarily in the D. melanogaster midline, including sog, shg, and NetA (Fig. 2A-2F) Similar to what was observed for sim, we found that these genes also have strong lateral CNS expression and no or severely diminished midline expression. Analysis of additional genes with broader CNS expression are also suggestive of a shift to more lateral regions (data not shown). Thus not just sim but a number of genes which in D. melanogaster are expressed in the midline have been redirected laterally and downregulated in the midline in A. aegypti. The fact that the expression of several genes involved in Drosophila midline development has shifted laterally in A. aegypti raises the intriguing possibility that what we are observing is the relocation of a gene regulatory network (GRN). In other words, a group of genes jointly involved in a developmental process may have redeployed as a unit to fulfill a new role, i.e., undergone neofunctionalization. Because the genes on which we have focused are best characterized with respect to their role in Drosophila midline development, and because the lateral shift has so far

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been observed only in A. aegypti, we will refer to the relevant GRN as the “midline GRN,” despite its apparent relocation away from the midline in A. aegypti.

CRMs for midline GRN genes from both species direct midline expression in D. melanogaster Redeployment of a GRN can arise from a series of cis-encoded changes affecting the cisregulatory modules (CRMs) of the GRN’s constituent genes, and/or from altered expression of one or more key transcription factors that regulate expression of GRN members (a transregulatory change). To gain insight into whether we are in fact witnessing GRN neofunctionalization, and if so, whether through cis- or trans-encoded mechanisms, we set out to characterize the D. melanogaster and A. aegypti GRNs by identifying relevant CRMs. We previously developed SCRMshaw, an effective method to guide CRM discovery in Drosophila (Kantorovitz et al., 2009; Kazemian et al., 2011b), and recently showed that this same method can be used in a cross-species manner to identify CRMs in more diverged insect species (Kazemian et al., 2014b). SCRMshaw uses a “training set” of known Drosophila CRMs, drawn from the REDfly database (Gallo et al., 2011), to develop a computational model that can be applied to CRM discovery in either D. melanogaster or another holometabolous insect. Because we lacked a sufficient training set of late midline stage CRMs, we used a mixed mesectoderm/midline primordium/late midline training set consisting of just eight validated CRMs (“mapping1.mesectoderm,” (Kazemian et al., 2014b)) to search both the D. melanogaster and A. aegypti genomes. Despite the limitations of our training set, we were able to predict CRMs from both species over the full range of midline developmental stages (based on recovery of all eight training set members and an additional 21 known D. melanogaster CRMs; see Table S1). We selected a total of 21 predicted CRMs — twelve in D. melanogaster and nine in A. aegypti – to test for activity using reporter gene assays in transgenic Drosophila (as described in (Kazemian et al., 2014b); Table S3). D. melanogaster candidates were selected from the top 200 ranked predictions, while A. aegypti candidates were further filtered for those which (1) mapped within 25 kb of a gene with a known D. melanogaster ortholog; (2) mapped within 25 kb of genes putatively in the midline GRN whose expression was judged to have shifted laterally in A. aegypti; and (3) for which we had a predicted CRM in both species (Table S2).

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Thirteen of the 21 (62%) candidate CRM sequences showed CRM activity in the transgenic fly assay (seven of the twelve D. melanogaster candidates (58%) and six of the nine (66%) predicted A. aegypti CRMs; Table S4; see below). This is substantially lower than our previous 75-100% successful prediction rate and may be a result of the small size of the training set and the temporal diversity of its member CRMs (Kantorovitz et al., 2009; Kazemian et al., 2014a; Kazemian et al., 2011b). Three CRMs drove gene expression at either the wrong stage (A. aegypti NetA_early_Aaeg and sim_early_Aaeg; Fig. S1) or in the wrong tissue (D. melanogaster CASK_A_Dmel; data not shown), and are not discussed further. Five predicted D. melanogaster CRMs (shg_A_Dmel, sema-1b_A_Dmel, Nox_A_Dmel, run_A_Dmel, and NetA_A_Dmel) drive CNS midline expression consistent with the native patterns of their associated genes (Fig. S2). However, as we have not so far identified A. aegypti CRMs for the same stages for the orthologs of these genes, these results do not yet shed light on the observed differences between the D. melanogaster and A. aegypti midline GRNs. The remaining D. melanogaster predicted CRM, sog_late_Dmel, drives sog-like midline expression starting at the midline primordium stage (Fig. 3C). For A. aegypti, we identified four midline GRN CRMs active in the targeted midline primordium and late midline stages, three for sog and one for Star. When tested in transgenic D. melanogaster, two of the A. aegypti sog CRMs (sog_750_Aaeg and sog2_Aaeg) recapitulate the expression of D. melanogaster sog in midline primordium cells (Fig. 3D; Fig. S3A), similar to the newly-predicted D. melanogaster sog_late_Dmel CRM. Interestingly, although consistent with D. melanogaster sog, the observed activity of the A. aegypti sog CRMs is contrary to the lateral CNS expression of A. aegypti sog (compare Fig. 2B with Fig. 3D). Reporter gene expression from two of these CRMs (A. aegypti sog_750_Aaeg and D. melanogaster sog_late_Dmel; Fig. S3B and data not shown) persists into the late midline stage. This is possibly due to perdurance of the LacZ reporter as D. melanogaster sog is not expressed in the midline or CNS after germ band retraction (>st 12) (Francois et al., 1994). The third A. aegypti sog CRM (sog3_Aaeg) regulates expression in the midline glia at the late midline stage only (Fig. S3C). This is consistent with the observed temporal expression profile, although not the lateral CNS location, of A. aegypti sog. Similar to the A. aegypti sog CRMs, an identified A. aegypti Star CRM, Star_F_Aaeg, shows activity in midline primordium cells in transgenic Drosophila (Fig. S3D) whereas endogenous A. aegypti Star, like the other A. aegypti genes, is predominantly expressed in the lateral CNS (Fig. S3F). These results, based on placing A. aegypti CRM reporters into a D. melanogaster genetic background, suggest that the midline vs. lateral gene expression differences observed between

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D. melanogaster and A. aegypti are due to a trans-regulatory, rather than a cis-regulatory, change. The single sim CRM (sim_early_Aaeg) identified in our SCRMshaw analysis regulates early gene expression only (Fig. S2B). However, because of Sim’s critical role as a key regulator of the midline GRN, we decided to search for additional A. aegypti sim CRMs by simply testing non-coding sequences from the sim locus for CRM activity. Surprisingly, we were largely unsuccessful in discovering embryonic sim CRMs. Despite testing over 30 kb of intronic and 5’ sequence (Fig. S5), we observed only minimal regulatory activity associated with just two large genomic fragments: a 3 kb fragment immediately 5’ to the annotated transcription start site (sim_up1_3kb_Aaeg) and an 8 kb fragment within the first intron of A. aegypti sim (sim_int1_P2_Aaeg). The activity of the former was manifest beginning at the midline primordium stage in paired midline glial cells (Fig. 3G, H) while the latter showed activity only at the late midline stage (~st. 13 onward; Fig. 3F). This expression, like that driven by the sog and Star CRMs (above), resembles the D. melanogaster midline pattern (Fig. 3E) more than the A. aegypti lateral pattern. The highly restricted pattern of reporter gene expression driven by these two large sequence fragments is surprising, and we cannot say with certainty at this time whether it represents a faithful recapitulation of low levels of sim expression in the A. aegypti midline, limited auto-regulatory activity due to Sim expression in the D. melanogaster midline, or a true trans-regulatory difference between the two species. Moreover, both the sim 5’_P1_Aaeg and sim_up2_3kb_Aaeg CRMs overlap with the sim_early_Aaeg CRM, yet did not produce any reporter gene activity. This is possibly due to the presence of repressor or insulator sequences within these larger fragments and needs further validation. Regardless, the restricted expression pattern, which is not faithful to the full range of sim expression in either D. melanogaster or A. aegypti at this stage, suggests that additional A. aegypti sim CRMs remain to be identified. Discovery of these additional CRMs will be essential in determining the mechanisms dictating the different patterns of sim expression in the two nervous systems.

Ectopic Sim expression alone is not sufficient to ectopically activate A. aegypti “midline” CRMs At the mesectoderm and midline primordium stages, Sim is a “master regulator” of Drosophila midline gene expression, sitting at the top of the midline GRN and directly activating the expression of many or possibly all of the downstream midline genes (Hong et al., 2013; Muralidhar et al., 1993; Nambu et al., 1991; Wharton et al., 1994), including those necessary for repressing genes involved in lateral CNS development (Estes et al., 2001). Toward the late 11

primordium stage and into the late midline stage, Sim’s role is more variable: while CRMs for some genes are strongly affected by loss of Sim binding, others show relatively minor effects (Pearson and Crews, 2014). This suggests that while Sim remains a necessary component of midline gene expression, at the later stages additional transcription factors (e.g., Su(H)) may also be playing important roles. While the mechanisms underlying lateral relocation of sim in A. aegypti must remain unknown pending identification of the relevant sim CRMs, an attractive hypothesis with respect to the rest of the midline GRN genes is that Sim is the trans-acting factor responsible for their lateral shift in A. aegypti. That is, loss of Sim from the A. aegypti midline could lead to loss of midline expression of downstream genes, while relocation of Sim to lateral CNS regions could direct their lateral expression. If this were the case, expression of Sim in the lateral Drosophila CNS should lead to ectopic activation of the A. aegypti CRMs. In order to test whether a change in the Sim expression pattern alone could account for the lateral relocation observed in the A. aegypti CNS, we expressed Sim in stripes in lateral CNS regions using prd-Gal4/UAS-sim (Pearson and Crews, 2014). Previous studies have shown that a number of Sim target genes, including rho, sog, and slit, as well as reporter gene expression driven by the rho-365 CRM, are induced in response to ectopic Sim CNS expression at the midline primordium stage (Nambu et al., 1991; Pearson and Crews, 2014; Zinzen et al., 2006). As expected, we found that the D. melanogaster sog_late_Dmel CRM drives laterally expanded expression in response to ectopic Sim at the late midline primordium stage and continuing into the late midline stage (Fig. 4B) (although the latter may simply be a result of lacZ perdurance). On the other hand, the D. melanogaster sema-1b_A__Dmel CRM failed to respond to ectopic Sim (Fig. 4D). This is consistent with the previous finding that Sim is not sufficient (although it may be necessary) to activate this CRM even in its wild-type midline location (Pearson and Crews, 2014). Similar to what was observed for the fly sema-1b_A_Dmel CRM, neither the A. aegypti sog750_Aaeg nor the A. aegypti Star_F_Aaeg CRMs responded to ectopic Sim (Fig. 4F and data not shown). Therefore, a simple shift in Sim expression to lateral CNS regions does not appear to be sufficient to explain the lateral shift of the midline GRN observed in A. aegypti. Signaling via the Notch pathway has also been suggested as a regulator of late midline gene expression (Pearson and Crews, 2014). We therefore tested if ectopic Notch activation could cause lateral expansion of reporter gene expression driven by the D. melanogaster and A. aegypti CRMs using prd-Gal4-mediated expression of the constitutively-signaling Notch intercellular domain. However, no expansion of reporter gene expression was observed (data

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not shown). It remains to be seen whether the combined activities of Sim and Notch are sufficient to expand expression off the midline.

Discussion SCRMshaw provides a powerful method for comparative GRN analysis across large evolutionary distances Comparative analysis of GRNs requires not only detailed gene expression data but crucially, knowledge of the relevant CRMs in both species under study. Given the challenges of CRM identification, this has often limited studies to closely-related species in which orthologous CRMs can be identified via sequence alignment (Gompel et al., 2005; Ordway et al., 2014; Prud'homme et al., 2006; Wittkopp et al., 2002). Because SCRMshaw can leverage Drosophila CRM data for CRM discovery across an extensive evolutionary range (Kazemian et al., 2014a), it is now possible to identify CRMs in parallel for the orthologous genes from D. melanogaster and much more distantly related insects in order to conduct detailed GRN comparisons. In this initial study we were able to identify CRMs in both D. melanogaster and A. aegypti for sim, sog, and NetA, although only for sog did we obtain truly equivalent CRMs (i.e., same gene, same stage) regulating gene expression at the late midline primordium and late midline stages of interest. However, we attribute the relatively low return in CRM discovery to the small and nonstage-specific training set used as input to SCRMshaw and expect that future iterations using more finely-tuned training data, which has become available in recent releases of the REDfly database, will greatly increase our yield of matched CRMs. While the limited set of equivalent CRMs precludes extensive sequence (motif) level analysis of the midline GRN CRMs at present, the several A. aegypti CRMs we identified have still enabled us to significantly advance our understanding of the molecular basis for the redeployed A. aegypti network. Resolving two additional issues will facilitate future more detailed investigations of this GRN. Because we are interested primarily in events during the late midline stage, we need to be certain that any observed CRM activity originates at that stage and does not merely represent reporter protein perdurance from an earlier timepoint. Using a rapid-turnover reporter (e.g., destabilized GFP, (Li et al., 1998)) or directly labeling nascent reporter gene RNAs (e.g. Pare et al., 2009) could help overcome this problem. Secondly, performing the reporter gene assays solely in Drosophila hinders the interpretation of results where trans-regulatory changes are in play and prevents a proper reciprocal analysis of CRMs from each species. Because

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transgenesis of A. aegypti is now possible via both transposon-mediated transformation (Coates et al., 1998; Jasinskiene et al., 1998) and CRISPR-Cas-mediated genome editing (Dong et al., 2015; Kistler et al., 2015), this issue can readily be addressed.

Trans-regulatory mediated GRN redeployment Our validated Drosophila CRMs showed activity in CNS midline cells consistent with the expression patterns of their presumed target genes. The four SCRMshaw-predicted Aedes aegypti CRMs active at late stages—three for sog and one for Star—also drove reporter gene expression in the midline, in stark contrast to the expression of the orthologous A. aegypti target genes. The fact that multiple A. aegypti CRMs for genes expressed laterally in the A. aegypti CNS all drove midline gene expression when placed in D. melanogaster strongly favors a transregulatory explanation for redeployment of the A. aegypti midline GRN. Definitive proof of this must await reciprocal reporter gene analysis in transgenic mosquitoes, where we predict that the D. melanogaster midline gene CRMs will drive the A. aegypti pattern of lateral expression. An obvious candidate for the chief trans regulator responsible for lateral relocation of the midline GRN in A. aegypti is Sim. Sim is a known regulator of this network, and sim expression in A. aegypti shifts laterally off the midline at the end of the midline primordium stage. Although ectopic expression of sim in transgenic flies did not result in a change in reporter gene activity mediated by the A. aegypti CRMs, similar experiments using Drosophila midline CRMs demonstrate that not all CRMs are under exclusive transcriptional control by Sim. While the sog CRM responds to ectopic sim expression, the sema-1b CRM does not. This is consistent with a previous study which found that multiple modes of regulation factor into late midline gene expression with Sim playing a necessary but not sufficient role (Pearson and Crews, 2014). Detailed analysis of our newly-discovered CRMs should yield insights into what additional regulators may be involved.

Regulation of sim In Drosophila, multiple CRMs control sim midline expression. Initial sim expression in the mesectoderm is controlled by an upstream 5’ CRM that contains binding sites for Dorsal, Twist, Snail and Suppressor of Hairless (Kasai et al., 1992; Markstein et al., 2004). This expression is maintained in the midline primordium from embryonic stage 9-11 by two separate autoregulatory CRMs containing Sim/Tango-binding sites (Muralidhar et al., 1993; Sonnenfeld et al., 1997;

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Sonnenfeld et al., 2005; Wharton et al., 1994). After stage 11, both of these CRMs are inactive, shutting down autoregulation-induced sim transcription (Freer et al., 2011; Nambu et al., 1991). Regulation of sim at this point in Drosophila development—the transition from the midline primordium to the late midline stage—is yet to be fully understood, although it has been proposed that a combination of Notch and Sim may regulate sim transcription (Pearson and Crews, 2014). It is at the comparable stage in Aedes aegypti development that we observe a change in the expression of sim from midline to lateral CNS. Thus it seems likely that the cis-regulatory mechanisms governing sim function up to this point in development, i.e., while sim is expressed in the midline, are conserved. However, confirmation of this must await identification of the relevant CRMs in A. aegypti. Beyond this stage, both the function and the means of regulation of sim are yet to be determined. We expect that here, the regulatory mechanisms between D. melanogaster and A. aegypti have diverged, making it unsurprising that our SCRMshaw analysis, which relies on conservation of regulatory strategies, failed to identify the A. aegypti late sim CRMs. However, our failure to discover additional sim CRMs even by “traditional” undirected reporter gene analysis is puzzling and suggests that a more in-depth analysis of the A. aegypti sim locus is required.

Functional consequences of GRN redeployment Our results demonstrate that Aedes aegypti has considerable differences in the expression patterns of numerous members of the Drosophila midline GRN during the late midline primordium and late midline stages of CNS development, despite an overall conservation in CNS morphology. Functional studies of midline development have so far not been carried out in A. aegypti for members of this GRN at any stage, and even in the more well-studied Drosophila, the details of the regulation and function of the midline GRN genes at these later-stage genes remain unknown. Thus in both species, and in A. aegypti in particular, extensive additional morphological, molecular, and functional characterization will be needed to determine the true extent of homology between the two species and the developmental and functional consequences of the changed gene expression. Knockdown studies of sim beyond the midline primordium stage in both species should shed light on the role of sim in the formation of the late midline, as will detailed analysis of additional members of the midline GRN. Also of interest will be to explore the expression and roles of A. aegypti genes whose D. melanogaster orthologs are expressed in the lateral CNS (e.g., ventral nervous system defective (vnd), intermediate

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neuroblast defective (ind), muscle segment homeobox (msh), etc.), to determine if lateral genes as well as midline genes have altered expression and/or function. A small subset of axon guidance genes—sema-1a, fra, comm2, and NetA/B—have been studied in detail in A. aegypti (Clemons et al., 2011; Haugen et al., 2011; Sarro et al., 2013) as well as in Drosophila (Brankatschk and Dickson, 2006; Georgiou and Tear, 2002; Harris et al., 1996; Kolodkin et al., 1993; Kolodziej et al., 1996; Mitchell et al., 1996; Tear et al., 1996). The expression patterns of these genes are mostly identical in the two species. However, siRNA knockdowns of A. aegypti sema-1a, fra, and comm2 revealed different severities of axonal phenotypes, with fra giving a more severe phenotype in A. aegypti than in Drosophila, sema-1a being more severe in some respects and less so in others, and comm2 being more severe in Drosophila (Clemons et al., 2011; Haugen et al., 2011; Sarro et al., 2013). These differences in midline development are subtle and reinforce the notion of essential similarity between the two species. At the same time, they are consistent with the notion that whereas in earlier stages the midline GRN is required for specifying midline cell fates, its later role—diverged between D. melanogaster and A. aegypti—may be more focused on discrete aspects of midline cell function, such as modulating the activity of midline neurons or regulating cell surface proteins necessary for mediating axon guidance.

Modes of GRN evolution Previous studies have described instances in which GRNs have been co-opted or repurposed through evolution, primarily in the context of morphological novelty (for review see (Rebeiz et al., 2015)). These GRN changes can be modest, causing limited phenotypic changes, such as the alterations in wing and body pigmentation in Drosophila caused by modification of bric-àbrac and yellow CRMs (Gompel et al., 2005; Williams et al., 2008). However, GRN changes can also have dramatic phenotypic consequences. For instance, mesoderm development in sea stars takes a different path than in sea urchins following initial specification of the mesoderm at the central vegetal pole of the embryo, but the same GRN subcircuit is directly upstream of both subsequent fates (McCauley et al., 2010). In each of these examples, a GRN has evolved “in place,” that is, a GRN acting in a common tissue has changed to yield a different downstream phenotype (Fig. 5A). The difference in scale between the fly and echinoderm examples results from the positioning of the GRN change, near the terminal nodes in the former and toward the top of the network in the latter.

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By contrast, Clark-Hachtel et al. have suggested that the carinated margin, a ridge on the body wall of the first thoracic segment in the beetle Tribolium castaneum, is a serial homolog of the wings in the second and third segments (Clark-Hachtel et al., 2013). These tissues appear to share a common wing-development GRN, including activity of a CRM for the important wingdevelopment gene nubbin. However, nubbin itself does not play a role in carinated margin development. In this case, therefore, the GRN remains intact in the second and third thoracic segments, but has evolved in a serially homologous location (the first thoracic segment) by altering its requirement for nubbin and potentially other, yet-to-be-defined factors (Fig. 5B). A similar scenario has recently been described in impressive detail in which the GRN governing development of the Drosophila posterior spiracle has been co-opted to generate a novel outgrowth of the male genitalia, the posterior lobe (Glassford et al., 2015). Unlike in the beetle carinated margin case, the posterior lobe is a completely novel structure rather than a serial homolog (Fig. 5C), and the events leading to redeployment of the spiracle GRN in the genitalia still need to be identified. Eye-spot patterning in the butterfly wing may represent a similar instance of a GRN being co-opted from a non-homologous structure. However, the relevant cistrans regulatory details have so far not been characterized, and the particular GRN that was coopted is the subject of debate (Monteiro, 2015). The GRN changes we report here represent a striking example of another, novel form of GRN evolution, one that helps to illustrate developmental system drift at the GRN level. The Drosophila midline GRN, which we take to be ancestral due to the persistent expression of sim into the late midline in the more basal holometabolous insects we examined, has undergone a “repeal, replace, and redeploy” mode of evolution in Aedes aegypti (Fig. 5D). The GRN is no longer active in its original midline territory but remains fully constituted in a new region, the lateral nerve cord, while presumably its original midline activity is now being carried out by a different, either co-opted or novel, GRN. Despite these many changes, overall nervous system morphology appears to be essentially unaltered, and observed phenotypic differences so far have been minor, providing a classic instance of developmental system drift and suggesting a remarkable degree of robustness in the face of significant genetic rewiring. While the causative basis for the GRN redeployment is not yet known, our analysis of CRMs from both species suggests changes in both cis- and trans-regulation. Future detailed studies in A. aegypti will be required to pin down the mechanistic details of the midline GRN replacement and neofunctionalization, and its full consequences.

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Acknowledgements We thank Steve Crews for Sim antiserum and for helpful comments on the manuscript, Yoshi Tomoyasu and Gene Robinson for generously providing T. cas and A. melanogaster embryos, respectively, Jeremy Lynch for sharing N. vit sim in situs, and Jack Leatherbarrow for support for fly genetics. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Funding for this work came from NIH grants R01 GM85233 (SS and MSH) and R01 AI081795 (MDS), and USDA grant 2012-67013-19361 (MSH and SS).

References Biffar, L., Stollewerk, A., 2014. Conservation and evolutionary modifications of neuroblast expression patterns in insects. Dev Biol 388, 103-116. Brand, A., Perrimon, N., 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415. Brankatschk, M., Dickson, B.J., 2006. Netrins guide Drosophila commissural axons at short range. Nat Neurosci 9, 188-194. Cande, J., Goltsev, Y., Levine, M.S., 2009. Conservation of enhancer location in divergent insects. Proc Natl Acad Sci U S A 106, 14414-14419. Carroll, S.B., Grenier, J.K., Weatherbee, S.D., 2005. From DNA to diversity : molecular genetics and the evolution of animal design, 2nd ed. Blackwell Pub., Malden, MA. Clark-Hachtel, C.M., Linz, D.M., Tomoyasu, Y., 2013. Insights into insect wing origin provided by functional analysis of vestigial in the red flour beetle, Tribolium castaneum. Proc Natl Acad Sci U S A 110, 16951-16956. Clemons, A., Haugen, M., Le, C., Mori, A., Tomchaney, M., Severson, D.W., Duman-Scheel, M., 2011. siRNA-mediated gene targeting in Aedes aegypti embryos reveals that frazzled regulates vector mosquito CNS development. PLoS One 6, e16730. Coates, C.J., Jasinskiene, N., Miyashiro, L., James, A.A., 1998. Mariner transposition and transformation of the yellow fever mosquito, Aedes aegypti. Proc Natl Acad Sci U S A 95, 3748-3751. Crews, S.T., Thomas, J.B., Goodman, C.S., 1988. The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product. Cell 52, 143-151. Davidson, E.H., 2006. The regulatory genome : gene regulatory networks in development and evolution. Academic, Burlington, MA ; San Diego. Dearden, P.K., Duncan, E.J., Wilson, M.J., 2009. Whole-mount in situ hybridization of honeybee (Apis mellifera) tissues. Cold Spring Harbor protocols 2009, pdb prot5225. Dong, S., Lin, J., Held, N.L., Clem, R.J., Passarelli, A.L., Franz, A.W., 2015. Heritable CRISPR/Cas9-mediated genome editing in the yellow fever mosquito, Aedes aegypti. PLoS One 10, e0122353. 18

Duman-Scheel, M., Patel, N.H., 1999. Analysis of molecular marker expression reveals neuronal homology in distantly related arthropods. Development 126, 2327-2334. Estes, P., Mosher, J., Crews, S.T., 2001. Drosophila single-minded represses gene transcription by activating the expression of repressive factors. Dev Biol 232, 157-175. Francois, V., Solloway, M., O'Neill, J.W., Emery, J., Bier, E., 1994. Dorsal-ventral patterning of the Drosophila embryo depends on a putative negative growth factor encoded by the short gastrulation gene. Genes & development 8, 2602-2616. Freer, S.M., Lau, D.C., Pearson, J.C., Talsky, K.B., Crews, S.T., 2011. Molecular and functional analysis of Drosophila single-minded larval central brain expression. Gene Expr Patterns 11, 533-546. Gallo, S.M., Gerrard, D.T., Miner, D., Simich, M., Des Soye, B., Bergman, C.M., Halfon, M.S., 2011. REDfly v3.0: toward a comprehensive database of transcriptional regulatory elements in Drosophila. Nucleic Acids Res 39, D118-123. Georgiou, M., Tear, G., 2002. Commissureless is required both in commissural neurones and midline cells for axon guidance across the midline. Development 129, 2947-2956. Glassford, W.J., Johnson, W.C., Dall, N.R., Smith, S.J., Liu, Y., Boll, W., Noll, M., Rebeiz, M., 2015. Co-option of an Ancestral Hox-Regulated Network Underlies a Recently Evolved Morphological Novelty. Developmental cell 34, 520-531. Gompel, N., Prud'homme, B., Wittkopp, P.J., Kassner, V.A., Carroll, S.B., 2005. Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila. Nature 433, 481-487. Harris, R., Sabatelli, L.M., Seeger, M.A., 1996. Guidance cues at the Drosophila CNS midline: identification and characterization of two Drosophila Netrin/UNC-6 homologs. Neuron 17, 217-228. Haugen, M., Flannery, E., Tomchaney, M., Mori, A., Behura, S.K., Severson, D.W., DumanScheel, M., 2011. Semaphorin-1a is required for Aedes aegypti embryonic nerve cord development. PLoS One 6, e21694. Hong, J.W., Park, K.W., Levine, M.S., 2013. Temporal regulation of single-minded target genes in the ventral midline of the Drosophila central nervous system. Developmental biology 380, 335-343. Housden, B.E., Millen, K., Bray, S.J., 2012. Drosophila Reporter Vectors Compatible with PhiC31 Integrase Transgenesis Techniques and Their Use to Generate New Notch Reporter Fly Lines. G3 2, 79-82. Jasinskiene, N., Coates, C.J., Benedict, M.Q., Cornel, A.J., Rafferty, C.S., James, A.A., Collins, F.H., 1998. Stable transformation of the yellow fever mosquito, Aedes aegypti, with the Hermes element from the housefly. Proc Natl Acad Sci U S A 95, 3743-3747. Kantorovitz, M.R., Kazemian, M., Kinston, S., Miranda-Saavedra, D., Zhu, Q., Robinson, G.E., Gottgens, B., Halfon, M.S., Sinha, S., 2009. Motif-blind, genome-wide discovery of cisregulatory modules in Drosophila and mouse. Dev Cell 17, 568-579. Kasai, Y., Nambu, J.R., Lieberman, P.M., Crews, S.T., 1992. Dorsal-ventral patterning in Drosophila: DNA binding of snail protein to the single-minded gene. Proceedings of the National Academy of Sciences of the United States of America 89, 3414-3418.

19

Kazemian, M., Suryamohan, K., Chen, J.-Y., Zhang, Y., Samee, M.A.H., Halfon, M.S., Sinha, S., 2014a. Evidence for Deep Regulatory Similarities in Early Developmental Programs across Highly Diverged Insects. Genome biology and evolution 6, 2301-2320. Kazemian, M., Suryamohan, K., Chen, J.Y., Zhang, Y., Samee, M.A., Halfon, M.S., Sinha, S., 2014b. Evidence for deep regulatory similarities in early developmental programs across highly diverged insects. Genome biology and evolution. Kazemian, M., Zhu, Q., Halfon, M.S., Sinha, S., 2011a. Improved accuracy of supervised CRM discovery with interpolated Markov models and cross-species comparison. Nucleic Acids Res. Kazemian, M., Zhu, Q., Halfon, M.S., Sinha, S., 2011b. Improved accuracy of supervised CRM discovery with interpolated Markov models and cross-species comparison. Nucleic acids research 39, 9463-9472. Kidd, S., Lieber, T., Young, M.W., 1998. Ligand-induced cleavage and regulation of nuclear entry of Notch in Drosophila melanogaster embryos. Genes & development 12, 3728-3740. Kistler, K.E., Vosshall, L.B., Matthews, B.J., 2015. Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti. Cell reports 11, 51-60. Kolodkin, A.L., Matthes, D.J., Goodman, C.S., 1993. The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 75, 1389-1399. Kolodziej, P.A., Timpe, L.C., Mitchell, K.J., Fried, S.R., Goodman, C.S., Jan, L.Y., Jan, Y.N., 1996. frazzled encodes a Drosophila member of the DCC immunoglobulin subfamily and is required for CNS and motor axon guidance. Cell 87, 197-204. Lewis, J.O., Crews, S.T., 1994. Genetic analysis of the Drosophila single-minded gene reveals a central nervous system influence on muscle development. Mechanisms of development 48, 81-91. Li, X., Zhao, X., Fang, Y., Jiang, X., Duong, T., Fan, C., Huang, C.C., Kain, S.R., 1998. Generation of destabilized green fluorescent protein as a transcription reporter. J Biol Chem 273, 34970-34975. Markstein, M., Zinzen, R., Markstein, P., Yee, K.P., Erives, A., Stathopoulos, A., Levine, M., 2004. A regulatory code for neurogenic gene expression in the Drosophila embryo. Development 131, 2387-2394. McCauley, B.S., Weideman, E.P., Hinman, V.F., 2010. A conserved gene regulatory network subcircuit drives different developmental fates in the vegetal pole of highly divergent echinoderm embryos. Developmental biology 340, 200-208. Mitchell, K.J., Doyle, J.L., Serafini, T., Kennedy, T.E., Tessier-Lavigne, M., Goodman, C.S., Dickson, B.J., 1996. Genetic analysis of Netrin genes in Drosophila: Netrins guide CNS commissural axons and peripheral motor axons. Neuron 17, 203-215. Monteiro, A., 2015. Origin, development, and evolution of butterfly eyespots. Annual review of entomology 60, 253-271. Muralidhar, M.G., Callahan, C.A., Thomas, J.B., 1993. Single-minded regulation of genes in the embryonic midline of the Drosophila central nervous system. Mechanisms of development 41, 129-138. Nambu, J.R., Franks, R.G., Hu, S., Crews, S.T., 1990. The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells. Cell 63, 63-75. 20

Nambu, J.R., Lewis, J.O., Wharton, K.A., Jr., Crews, S.T., 1991. The Drosophila single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development. Cell 67, 1157-1167. Oda, H., Uemura, T., Harada, Y., Iwai, Y., Takeichi, M., 1994. A Drosophila homolog of cadherin associated with armadillo and essential for embryonic cell-cell adhesion. Dev Biol 165, 716726. Ordway, A.J., Hancuch, K.N., Johnson, W., Wiliams, T.M., Rebeiz, M., 2014. The expansion of body coloration involves coordinated evolution in cis and trans within the pigmentation regulatory network of Drosophila prostipennis. Developmental biology 392, 431-440. Pare, A., Lemons, D., Kosman, D., Beaver, W., Freund, Y., McGinnis, W., 2009. Visualization of individual Scr mRNAs during Drosophila embryogenesis yields evidence for transcriptional bursting. Curr Biol 19, 2037-2042. Pearson, J.C., Crews, S.T., 2014. Enhancer diversity and the control of a simple pattern of Drosophila CNS midline cell expression. Developmental biology 392, 466-482. Peter, I.S., Davidson, E.H., 2011. Evolution of gene regulatory networks controlling body plan development. Cell 144, 970-985. Prud'homme, B., Gompel, N., Rokas, A., Kassner, V.A., Williams, T.M., Yeh, S.D., True, J.R., Carroll, S.B., 2006. Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene. Nature 440, 1050-1053. Prud'homme, B., Minervino, C., Hocine, M., Cande, J.D., Aouane, A., Dufour, H.D., Kassner, V.A., Gompel, N., 2011. Body plan innovation in treehoppers through the evolution of an extra wing-like appendage. Nature 473, 83-86. Rebeiz, M., Jikomes, N., Kassner, V.A., Carroll, S.B., 2011. Evolutionary origin of a novel gene expression pattern through co-option of the latent activities of existing regulatory sequences. Proc Natl Acad Sci U S A 108, 10036-10043. Rebeiz, M., Patel, N.H., Hinman, V.F., 2015. Unraveling the Tangled Skein: The Evolution of Transcriptional Regulatory Networks in Development. Annual review of genomics and human genetics 16, 103-131. Sarro, J., Andrews, E., Sun, L., Behura, S.K., Tan, J.C., Zeng, E., Severson, D.W., DumanScheel, M., 2013. Requirement for commissureless2 function during dipteran insect nerve cord development. Dev Dyn 242, 1466-1477. Schinko, J., Posnien, N., Kittelmann, S., Koniszewski, N., Bucher, G., 2009. Single and double whole-mount in situ hybridization in red flour beetle (Tribolium) embryos. Cold Spring Harbor protocols 2009, pdb prot5258. Simanton, W., Clark, S., Clemons, A., Jacowski, C., Farrell-VanZomeren, A., Beach, P., Browne, W.E., Duman-Scheel, M., 2009. Conservation of arthropod midline netrin accumulation revealed with a cross-reactive antibody provides evidence for midline cell homology. Evol Dev 11, 260-268. Sonnenfeld, M., Ward, M., Nystrom, G., Mosher, J., Stahl, S., Crews, S., 1997. The Drosophila tango gene encodes a bHLH-PAS protein that is orthologous to mammalian Arnt and controls CNS midline and tracheal development. Development 124, 4571-4582. Sonnenfeld, M.J., Delvecchio, C., Sun, X., 2005. Analysis of the transcriptional activation domain of the Drosophila tango bHLH-PAS transcription factor. Development genes and evolution 215, 221-229.

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Tautz, D., Pfeifle, C., 1989. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85. Tear, G., Harris, R., Sutaria, S., Kilomanski, K., Goodman, C.S., Seeger, M.A., 1996. commissureless controls growth cone guidance across the CNS midline in Drosophila and encodes a novel membrane protein. Neuron 16, 501-514. Tessier-Lavigne, M., Goodman, C.S., 1996. The molecular biology of axon guidance. Science 274, 1123-1133. True, J.R., Haag, E.S., 2001. Developmental system drift and flexibility in evolutionary trajectories. Evol Dev 3, 109-119. Weiss, K.M., Fullerton, S.M., 2000. Phenogenetic drift and the evolution of genotype-phenotype relationships. Theoretical population biology 57, 187-195. Wharton, K.A., Jr., Franks, R.G., Kasai, Y., Crews, S.T., 1994. Control of CNS midline transcription by asymmetric E-box-like elements: similarity to xenobiotic responsive regulation. Development 120, 3563-3569. Wheeler, S.R., Kearney, J.B., Guardiola, A.R., Crews, S.T., 2006. Single-cell mapping of neural and glial gene expression in the developing Drosophila CNS midline cells. Developmental biology 294, 509-524. Wheeler, S.R., Stagg, S.B., Crews, S.T., 2008. Multiple Notch signaling events control Drosophila CNS midline neurogenesis, gliogenesis and neuronal identity. Development 135, 3071-3079. Williams, T.M., Selegue, J.E., Werner, T., Gompel, N., Kopp, A., Carroll, S.B., 2008. The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila. Cell 134, 610-623. Wittkopp, P.J., Vaccaro, K., Carroll, S.B., 2002. Evolution of yellow gene regulation and pigmentation in Drosophila. Current biology : CB 12, 1547-1556. Xiao, H., Hrdlicka, L.A., Nambu, J.R., 1996. Alternate functions of the single-minded and rhomboid genes in development of the Drosophila ventral neuroectoderm. Mechanisms of development 58, 65-74. Zinzen, R.P., Cande, J., Ronshaugen, M., Papatsenko, D., Levine, M., 2006. Evolution of the ventral midline in insect embryos. Dev Cell 11, 895-902. Figure 1: The A. aegypti sim expression pattern has diverged in the late midline. In situ hybridizations showing the expression of sim in three diverged insect species – D. melanogaster (A-D), A. mellifera (E-G) and A. aegypti (H, I) at two different stages in embryogenesis. (A, B) Ventral views of whole embryos, anterior is to the left. Boxes indicate representative regions magnified in the remaining panels, which show approximately four segments each. Arrowheads in B point to mesodermal sim expression in muscle cells lateral and ventral to the CNS. (A, C) At the midline primordium stage in D. melanogaster (stage 11), sim is expressed throughout the ventral midline and in repeated, segmental clusters of mesodermal muscle progenitor cells (arrowheads in C). (D) At the late midline stage in D. melanogaster (stage 15), sim expression is 22

confined to a subset of midline cells. (E) The A. melanogaster sim expression pattern is similar to that of D. melanogaster, including the lateral clusters which we take to be muscle progenitors (arrowheads). (F) A. melanogaster sim expression persists in the midline and can be observed in developing ventral oblique muscle fibers (arrowhead). The close-up in (G) at a later stage clearly shows that cells with the more lateral expression have the morphology of muscle fibers (arrowhead) and lie beyond the edge of the CNS (arrow). An asterisk marks the midline expression toward the top of the panel. (H) A. aegypti sim is expressed in the midline through the midline primordium stage (pictured), but shifts laterally at the late midline stage (I) with little or no midline expression remaining. (J) sim persists in the late midline in the mosquito Culex quinquefasciatus and also (K) in the jewel wasp Nasonia vitripennis (Nasonia image courtesy Jeremy Lynch). Figure 2: D. melanogaster midline genes are expressed laterally in the A. aegypti CNS. Expression of genes that are expressed in the D. melanogaster midline at the midline primordium and late midline stages, in D. melanogaster (A, C, E) and A. aegypti (B, D, F). All panels show ventral views of 4-5 segments of each embryo with anterior to the left. Data are from whole mount in situ hybridization with the exception of panel C, which shows protein expression from immunocytochemistry. In each panel, the midline is marked with an arrowhead and small arrows indicate the approximate boundary of the CNS. Overall, for all genes, midline expression in the A. aegypti embryos is weak-to-absent. (A) sog is strongly expressed in the D. melanogaster midline through stage 11. (B) In contrast, A. aegypti sog is expressed in the lateral CNS at a comparable stage (43-44 hours). Unlike for D. melanogaster, in which sog expression begins in the mesectoderm and converges to the midline at early germband extension, there is no early A. aegypti sog expression in the developing CNS (data not shown). (C) At the late midline stage, D. melanogaster shg is observed in the CNS solely in the midline, significantly different from the primarily lateral expression seen for A. aegypti shg (D). (E) NetA is expressed in the D. melanogaster ventral midline at the late midline stage (stage 15) in a subset of midline cells. (F) A. aegypti NetA expression is seen in the lateral CNS at the equivalent stage (50-52 hrs). Panel C adapted from (Oda et al., 1994), used with permission. Figure 3: Experimental validation of SCRMshaw-predicted D. melanogaster and A. aegypti midline gene CRMs. (A, B) Cartoon of expression patterns of D. melanogaster midline genes (A) and their respective orthologs in A. aegypti (B). (C-H) Predicted CRMs from A. aegypti and D. melanogaster were tested in transgenic D. melanogaster. (C) The D. melanogaster sog_late CRM drives reporter gene expression (green) that resembles the known sog midline expression

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pattern. Endogenous sog is expressed in the midline coincident with Sim, shown here in magenta (overlap is white). (D) The predicted A. aegypti sog_750 CRM also drives a midlinespecific reporter gene expression, similar to that of the D. melanogaster sog_late CRM (C) but in stark contrast to the expected activity based on the observed A. aegypti sog pattern (B) (compare panel D with Fig. 2B). (F) The A. aegypti sim_int1_P2 CRM, from within the first intron of A. aegypti sim, drives reporter gene expression in the late midline in a subset of midline glial cells in partial recapitulation of the known expression pattern of D. melanogaster sim (E), again in contrast to the expected observed endogenous A. aegypti pattern (B). (G, H) Similarly, the sim_up1_3kb fragment from the A. aegypti sim upstream region drives midline expression in D. melanogaster embryos at both the midline primordium (G) and late midline (H) stages, instead of in the expected lateral CNS location (compare panels F-H with Fig. 1I). A white arrowhead marks the midline in panel H. All panels show ventral views with anterior to the left. Figure 4: Effect of ectopic Sim expression on identified A. aegypti and D. melanogaster CRMs. Transgenic embryos with D. melanogaster or A. aegypti CRM-driven reporter genes were stained at the midline primordium stage for LacZ expression in the presence (B, D, F) or absence (A, C, E) of ectopic Sim driven by prd-GAL4. Magenta staining in (D) shows the boundaries of the ectopic Sim expression. The D. melanogaster sog_late CRM responds to ectopic Sim expression (compare panels A and B), but the D. melanogaster sema-1b_A CRM does not (C, D). Like the D. melanogaster sema-1b_A CRM, the A. aegypti sog750 CRM is nonresponsive to ectopic Sim (E, F). Embryos are ventral up and anterior to the left at either stage 10 (C-F) or 11 (A, B). Figure 5: Modes of GRN evolution. Examples of at least four different modes of GRN evolution have been described to varying degrees of detail. Each panel depicts a set of “cells” (gray circles) organized into three “segments” (black rectangles) in a hypothetical embryo. (A) GRN changes can alter the functional output of a GRN (red) to produce a new phenotype “in place” in a directly homologous tissue or organ (blue), in lieu of the ancestral phenotype. (B, C) Alternatively, the ancestral function and phenotypes can be maintained (red) while the GRN is simultaneously co-opted to produce either an altered serial homolog of the original structure (B), or a novel non-homologous structure or phenotype (C) in the evolved species (blue). (D) In this study, we have demonstrated a fourth example of GRN evolution whereby the GRN has been co-opted to a non-homologous territory in the evolved species (blue) and is no longer performing its ancestral function (red). The original function may either be lost altogether or replaced by another GRN (purple).

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Highlights    

Genes expressed in the Drosophila CNS midline shift laterally in Aedes aegypti This suggests redeployment of a gene regulatory network (GRN) Analysis of reporter genes suggests that trans-dependent mechanisms predominate We report a novel mode of GRN evolution mediating developmental system drift

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